Construction Of Food-grade Expression System Based On GlmS Gene Selection Marker In Lactobacillus

In this study,the Cre-lox system was used to delete the target gene.Firstly,bsh gene was deleted on the chromosome of Lactobacillus plantarum WCFS1.The plasmid pXU003 which carried the bsh gene upstream and downstream about 1 kb homologous arm was successfully constructed.Then the electrotransformation conditions of Lactobacillus plantarum was studied by optimizaing four aspects:competent cell preparation method,plasmid concentration,voltage,cell recovery time.The best electrotransformation conditions were established.The best competent cell preparation method is PEG method.The best plasmid amout is 4?L extracted by TB(plasmid concentration is about 1.6?g/?L).The best voltage is 2200v.The best recovery time is 2h.This study improved the electrotransformation efficiency of the plasmid to Lactobacillus plantarum significantly.It also solved the problem of low homologous recombination efficiency of gene knock-out.The Successful knock-out of the bsh gene in WCFS1 provided beneficial conditions for the knock-out of the glmS gene.On the basis of this,this study constructed the knock-out plasmid pXU004 of suceessfully.glmS gene was deleted on the chromosome of Lactobacillus plantarum WCFS1 successfully and the resistance gene selection marker was removed by Cre-lox system.The achievement of WCFS1-?glmS strain is a necessary condition of the food-grade expression system construction.Subsequently,this study constructed plasmid pHIS460 carrying his-tag and expressed glmS.Western-blot result showed that recombinant GlmS-His protein was successful.It is another necessary condition of the food-grade expression system construction.To sum up,this study provided beneficial conditions for the expression system of WCFS1 based on glmS gene selection marker.It also provided theoretical basis for the construction of a food-grade expression system of lactobacillus.